1a.Objectives (from AD-416)
The overall objective of this project is to define the minimal sets of genes required for efficient synthesis and accumulation of industrially important fatty acids in transgenic hosts, and to express these genes in microbes and commodity oilseed crops for production of value-added industrial oils. During the project, we will focus on the following objectives:
Objective 1: Use model plant systems to identify and refine transgenic expression conditions for critical industrial oil biosynthetic genes.

Objective 5: Engineer tung FADX, DGAT2, and other genes from donating organism (tung tree) into commercially important oilseed crop plant such as cotton, soybean, or camelina.

1b.Approach (from AD-416)
Genes encoding the enzymes for tung oil biosynthesis will be identified by homology-based searches and next-generation high volume pyrosequencing technologies. Other necessary enzymes and proteins will be detected via transcriptomic and proteomic analysis of seeds from tung and other species. Comparisons between different species of tung that produce medium or high amounts of eleostearic will also be used to detect evolution of enzymes well-suited to tung oil production. Mutagenesis studies will identify the active sites and critical residues in these enzymes, thus facilitating the design of engineered forms of important proteins. Model laboratory species of plants and microbes will be used to express combinations of multiple tung genes to find the minimal sets necessary to produce useful levels of eleostearic and other novel fatty acids. A microbial expression system tailored for the bioconversion of low-cost oils into tung-like drying oils will be generated by engineering common yeast strains to efficiently use oils as food, convert the common fatty acids to new valuable lipids, and increase the cellular lipid content.

3.Progress Report
This year, the tung FADX gene was expressed in transgenic plants using five different promoters and ranked based on the average level of the finishing oil, eleostearic acid (the product of the FADX enzyme), produced in the transgenic seed oils.

Eleostearic acid is produced in one part of the cells of developing seeds, but packaged into oil at a different site. One limitation to novel fatty acid production is an unknown biochemical blockage between the cellular sites where fatty acids are modified and where they are packaged into oil. One of the challenges to plant oilseed metabolic engineering is to determine which enzymes are necessary to drive efficient movement of the novel fatty acids to the site of oil packaging. Three additional enzymes (or proteins) [choline phosphotransferase (CPT), lysophosphatidylcholine acyltransferase (LPCAT), and phospholipid:diacylglycerol acyltransferase (PDAT)] which are thought to act in between these two sites, are currently being tested.

Most oil synthetic enzymes are targeted to cellular membranes, making them difficult to purify for further study. Tung tree DGAT1 and DGAT2 enzymes were fused to other proteins that are known to increase solubility. When expressed in bacteria, a portion of these fused proteins remained soluble and were partially purified. These studies established the first procedures for expressing full-length DGAT proteins from any species using a bacterial expression system. Recombinant proteins will be further purified for antibody production.

LPAT enzyme activity is known to play an important role in determining the fatty acid composition of several important vegetable oils, such as coconut and canola. Plants contain large families of LPAT genes. To date, six potential LPAT genes have been isolated from tung tree. Paired with the tung DGAT2 gene (which is known to be important to tung oil synthesis), each of the six potential tung LPAT genes has been built into baker’s yeast lines, and will be analyzed for increased production of tung-like drying oils.

Yeasts were modified to produce a self-controlling lipase enzyme (which is an enzyme that breaks down oil so that the yeast can consume it). This lipase is only produced when oils and other complex lipids are present outside the yeast cells. This is an important step in engineering of yeasts, because they are otherwise unable to take up complex lipids from their habitat. Lipase production will assist in conversion of baker’s yeast into a form of “bioreactor” that can grow on low-value waste lipids or commodity vegetable oils to produce various value-added lipids, including tung-like drying oils.

Many proteins and enzymes are chemically modified after they are produced in cells. These modifications help to control the activity of enzymes. Previous studies suggest addition of phosphate may increase enzyme activity. The tung DGAT enzymes were expressed in different types of microbes. Some data suggested that the nature of DGAT enzymes produced in bacteria (which cannot carry out chemical modifications) is different compared to those made in yeast, which can modify proteins.

4.Accomplishments
1.
Production of inducible, secretable lipase system in baker’s yeast. Yeasts and other microbes are a viable alternative to oilseed plants as a host for production of industrial oils and other value-added lipids. However, yeasts cannot take up complex oils directly from their surroundings. ARS researchers in the Commodity Utilization Research Unit, in New Orleans, LA, produced a genetic system that allows yeast to secrete a self-regulating lipase enzyme only when oils are present. This is an important step in metabolic engineering of transgenic yeasts for production of tung-like drying oils and other industrial oils, and will ultimately assist in conversion of baker’s yeast into a safe biorefinery system.

2.
First transgenic soluble expression of full-length DGAT enzymes from any system. Detailed characterization of important oil synthesizing enzymes, like tung DGAT1 and DGAT2, has been slowed by an inability to produce large amounts of these enzymes in a soluble form. ARS researchers in the Commodity Utilization Research Unit, in New Orleans, LA, coupled the proteins for tung DGAT1 and DGAT2 to another protein, called maltose binding protein. The hybrid proteins were produced in a soluble form in a bacterial expression system, which has never been accomplished before. The ability to produce soluble forms of these enzymes will assist in future efforts to carry out studies of the structure and function of plant DGATs, and to raise antibodies against them for other types of analysis. Ultimately, these results will assist in refining and developing strategies for production of high-performing transgenic industrial oilseed crops.